Cell, Volume 130

Supplemental Data

Mec1/Tel1 Phosphorylation of the INO80

Chromatin Remodeling Complex Influences

DNA Damage Checkpoint Responses Ashby J. Morrison, Jung-Ae Kim, Maria D. Person, Jessica Highland, Jing Xiao, Tammy S. Wehr, Sean Hensley, Yunhe Bao, Jianjun Shen, Sean R. Collins, Jonathan S. Weissman, Jeff Delrow, Nevan J. Krogan, James E. Haber, and Xuetong Shen

Figure S1. INO80 in vitro ATPase activity is altered in mec1 tel1 mutant cells. INO80 complexes were purified with 0.5 M KCl buffer from wild-type BY4705 (Wt) or mec1

tel1

that were untreated or treated with 0.25% MMS (+MMS) for three hours. Equal amounts of each purified complex were used in multiple ATPase assays stimulated by nucleosomes (Nuc.) or DNA. Autoradiograms of hydrolyzed 32P-ATP were quantitated and the average relative hydrolysis is shown with the untreated sample of each strain set to 1. Error bars denote standard deviation among replicate samples from independent experiments.

Figure S2. ies4 phosphorylation mutants do not alter binding of the complex to DNA damage sites. ChIP experiments that measure the recruitment of Ino80 and Ies4 to a DSB. JKM179 strains were grown for two hours in media containing either 2% glucose (- Gal) to repress the HO endonuclease or 2% galactose (+ Gal) to induce the HO endonuclease. (A) The strains used include IES4 deletion strains (ies4 ) containing chromosomally FLAG-tagged INO80 (Ino80-F), as well as plasmids encoding either wild-type IES4 or the ies4 S:E-5 phosphorylation mutant described in Figure 4A. PCR primers amplify regions +1 and +3 kilobases (kb) from DSB. (B) ChIP analysis of strains lacking the IES4 gene (ies4 ) and containing a plasmid encoding FLAG-tagged IES4 (Ies4-F) with either the wild-type IES4 sequence or the ies4 S:E-5 phosphorylation mutant described in Figure 4A are shown. PCR primers amplify a region +1 kilobase (kb) from DSB. Real-time PCR results are an average of two independent experiments and are shown as “Relative Fold Enrichment” above background levels. All "+ Gal" samples have at least 90% DSB at the MAT locus.

Figure S3. Mutation of Ies4’s phosphorylation sites does not significantly affect transcription. (A) Serial dilutions (1:5) of wild-type (Wt) BY4741 strain or an IES4 deletion strain (ies4 ) carrying either empty vector (vector), a vector containing the wild-type IES4 gene (IES4), or ies4 phosphorylation mutants (ies4), as described in Figure 4A, were plated on either normal media (YPD) or media lacking inositol (-Inositol). ino80

is shown as a control. (B) Venn diagrams illustrate the results from microarray analyses comparing the transcription expression profiles of wild-type cells and, either an INO80 deletion strain (ino80 ), or a strain lacking the wild-type IES4 gene and carrying a plasmid containing the ies4 S:E-5 mutations described in Figure 4A.

Figure S4. ies4 phosphorylation mutants do not affect non-homologous end-joining or strand invasion/primer extension, ligation, or viability during homologous recombination. (A) The DSB at the MAT locus is repaired via non-homologous end-joining. Serial dilutions (1:5) of JKM179 strains containing the genetic alterations described in Figure 4A. Strains were grown in media containing either 2% glucose or galactose in order to repress (- HO) or induce (+ HO) expression of the HO endonuclease. A yku80 strain is shown as a control. (B) Diagram depicts the strand invasion/primer extension step during homologous recombination. Arrows denote location of primers. YFP17 strains that lack IES4 (ies4 ) and carry ies4 phosphorylation mutants, as described in Figure 4A, were grown in galactose-containing media for the indicated time in hours (hrs) to induce expression of the HO

endonuclease. PCR results are shown on EtBr stained gels (middle panels). The graph displays the quantified signal of the PCR (bottom panel) normalized by the PCR amplified CEN8 sequence for each strain (data not shown). (C) YMV80 strains that repair a DSB at the MAT locus with single-strand annealing and carry the genetic manipulations described in Figure 4A were grown in galactose-containing media for the indicated times to induce expression of the HO endonuclease (+HO). Real-time PCR results are shown in bottom panel that monitor the formation of the ligated repair product and normalized using the real-time PCR signal obtained from the CDC13 locus. All samples had at least 95% of cells containing a DSB, as determined by real-time PCR using primers annealing to DNA on both sides of the DSB. (D) YFP17 strains lack IES4 (ies4 ) and carry ies4 phosphorylation mutants as described in Figure 4A. The viability of strains was determined by dividing the number of colonies growing in galactose-containing media (+HO) by the number of colonies growing in glucose-containing media (-HO). In all experiments shown in Supplemental Figure 4, plasmids were stably maintained by growing strains on SC -ura media.

Figure S5. ies4 phosphorylation mutants do not influence the rate of product formation during homologous recombination. (A) Diagram shows the HO-induced DSB at the MAT locus and the donor sequence used during homologous recombination. Location of the probe used for Southern blotting and products of EcoRI digest are shown. (B) Strains described in Supplemental Figure 4B were grown in galactose-containing media for the indicated time in hours (hrs) to induce expression of the HO endonuclease. Results of Southern blotting are shown with the single asterisk (*) denoting the donor sequence and the double asterisk (**) denoting the major repair product. The graph displays the quantified and normalized signal of the major repair product for each strain. Plasmids were stably maintained by growing strains in SC -ura media.

Figure S6. Rad53 phosphorylation is elevated in cells that mimic persistently phosphorylated Ies4. Strains described in Figure 4A were grown to mid-log phase in SC -ura media and treated with 0.045% MMS for the indicated times. Rad53 western blots are shown with the coomassie stained gel shown as a loading control (Load).

Figure S7. Mutations that alter Ies4 phosphorylation do not affect checkpoint recovery. (A) Strains described in Figure 4A were grown to mid-log phase in SC -ura media and either untreated (-MMS) or treated with 0.045% MMS for two hours (+MMS). The cells were then washed repeatedly and allowed to resume growth in media lacking MMS (Hours Post-MMS). Rad53 and -H2AX westerns are shown with the corresponding coomassie stained gel shown as a loading control (Load). (B) Serial dilutions (1:5) of strains, described in Supplemental Figure 7A, before (-MMS) and after MMS treatment (+MMS) were grown on SC -ura media without MMS.

Figure S8. ies4 phosphorylation mutants do not alter the association of -H2AX with the INO80 complex. (A) INO80 complexes were purified with 0.25 M KCl buffer from strains described in Figure 4A that were treated with 0.25% MMS for two hours. Purified samples were treated with RNase A and DNase 1 and electrophoresed on SDS-PAGE gels. Silver stain and -H2AX western of purified INO80 complexes is shown (Purified Protein), along with -H2AX and coomassie stain of whole cell extracts (Total Protein). The deletion of IES4 is not obvious in this experiment because it comigrates with Ies5 and Ies6 in high percentage SDS-PAGE gels.

Figure S9. A mutant that mimics phosphorylated Ies4 genetically interacts with genes in checkpoint pathways and not with DNA repair genes. Scatter plot results of SGA analysis using E-MAP technology are shown. Each spot represents a double mutant containing a gene listed in Supplemental Spreadsheet 1, as well as ies4 phosphorylation mutations described in Figure 4A (X and Y axes). Viability of double mutants is shown with synthetic lethal (or sick) mutants scored negatively and synthetic rescued mutants scored positively. Genes that score similarly in both ies4 phosphorylation mutants appear along diagonal line. Both ies4 phosphorylation mutants have similar genetic interactions with each other (Pearson correlation coefficient = 0.81). Several cell cycle checkpoint genes are highlighted in red and DNA repair genes are highlighted in blue.

Figure S10. A mutant that mimics phosphorylated Ies4 is epistatic to RAD54. (A) Serial dilutions (1:5) of wild-type (Wt), ies4 and rad54 single and double deletion strains carrying either an empty vector (vector), a vector containing the wild-type IES4 gene (IES4), or ies4 phosphorylation mutants, as described in Figure 4A. Strains were plated on either normal media (No treatment), media containing 0.01% MMS, or 50 mM HU. Plasmids were stably maintained by growing strains in SC -ura media.

Supplemental Experimental Procedures

In vitro ATPase Assays Nucleosome-stimulated ATPase assays were performed as previously described (Shen et al., 2003). Where noted DNA was used to stimulate activity instead of nucleosomes (Panyutin et al., 1995). Wild-type BY4705 and the previously described mec1

tel1

strain

were used in these assays (Chan et al., 2001).

Protein Purification and Chromatin Immunoprecipitation (ChIP) Purification of INO80 complexes were performed as previously described (Morrison et al., 2004; Shen, 2004). For ChIP experiments, INO80 was chromosomally-tagged with a triple FLAG sequence in JKM179 cells by using a PCR-mediated method with the p3FLAG-KanMX as a template, as previously described (Mizuguchi et al., 2004). IES4 was cloned with its natural promoter into the pRS416 plasmid and transformed into JKM179 strain containing a chromosomal FLAG-tagged INO80. FLAG-tagged IES4 was constructed as described in main text. ChIP experiments were performed essentially as described, with a few modifications (Burke et al., 2000). Cells expressing FLAG-tagged proteins were crosslinked with 1% formaldehyde for 15 minutes at 30 C. Real-time PCR results for each sample were normalized using DNA from the corresponding input sample. The non-specific background level, which was determined by performing ChIP analysis on yeast strains lacking FLAG-tagged proteins, was subtracted from each sample. Primers used to determine the recruitment of proteins to the MAT locus, as well as the procedure to determine the percentage of cells that contain a double strand break at the MAT locus, was determined by real-time PCR as previously described (Morrison et al., 2004).

Expression Profiling and Array Data Analysis Microarray construction and hybridization protocols are described elsewhere (Fazzio et al., 2001). A description of the yeast ORF microarray used in this study can be found in the NCBI Gene Expression Omnibus (GEO) repository – platform accession number GPL1914. Post-hybridized arrays were scanned using a GenePix 4000B scanner (Molecular Devices Corp., Sunnyvale, CA) and the subsequent image analysis was performed using GenePix Pro 6.0 software.

For each array, spot-level intensity signals were filtered and removed from further analysis if the signal-to-noise ratio was less than 3 in both channels or if the spot was assigned a ‘bad’ flag by the GenePix Pro software. The signal from each spot was corrected to remove local background and log2(cy5/cy3) values were calculated. Intra-array normalization was performed using a lowess algorithm to correct for intensity-dependent ratio biasing (Yang et al., 2002). Statistical data analysis was performed using CyberT (Baldi and Long, 2001), a Bayesian t-statistic derived for microarray analysis. Changes in relative expression were identified as significant by ranking the Bayesian p-values and applying a false discovery rate (FDR) algorithm to account for multiple testing (Benjamini and Hochberg, 1995). A 1.5 fold threshold was used in these analyses. Therefore, the number of genes regulated by INO80 is smaller compared to a previous study using a 1.25 fold threshold (Mizuguchi et al., 2004). .

Homologous Recombination (HR) and Non-homologous end-joining (NHEJ) Assays For NHEJ experiments, the JKM179 strain described in main text was used. For HR experiments, the YFP17 strain carrying a galactose inducible HO-endonuclease and an ectopic donor sequence for homologous recombination of a double-strand break (DSB) at the MAT locus was transformed with pRS416 plasmids encoding Ies4 proteins (Brachmann et al., 1998; Vaze et al., 2002). For HR viability assays, colonies were counted three days after diluted strains were plated on media containing either glucose or galactose. For ligation assays during single-strand annealing, strains derived from the YMV80 strain carrying a galactose-inducible HO endonuclease were grown in galactose-containing media (Vaze et al., 2002). Real-time PCR measuring DSB creation and repair was performed using primers previously described, except for SSA P1, which is 5’-TCTGGCTGGAGGTCACCAACGTCAA-3’(Keogh et al., 2006). For HR strand invasion and product formation experiments, galactose was added to a final concentration of 2% DNA was purified from cells at the indicated timepoints. Primers used to assay for strand invasion and primer extension are: forward, 5’-AAAGAAGAAGTTGCAAAGAAATGTGG-3’; reverse, 5’-GTGTTCCCGATTCAGTTTGACG-3’. For Southern blot assays, genomic DNA was digested with EcoRI and probed with a PCR amplified fragment using the primers: forward, 5’-ATGCCTGACTTCTTTTGACGAGG-3’; reverse, 5’-CCGCATGGGCAGTTTACCT-3’.

Protein detection and E-MAP analysis Western blots and total protein staining were performed as described in main text. E-MAP analysis was performed as described in main text.

Strains, Plasmids and Epitope-tagging Procedures Construction of the BY4733 strain encoding INO80 with a chromosomal double FLAG tag was previously described (Shen, 2004). INO80 and IES4 were cloned into pRS415 (LEU2) or pRS416 (URA3) vectors containing a double FLAG sequence, as previously described (Shen et al., 2003). INO80 was chromosomally FLAG-tagged in the BY4705 mec1

tel1

strain by transformation using a PCR fragment containing the C-terminus of INO80 with a double FLAG sequence and URA3 marker. The template for this PCR fragment was DNA from a strain that was transformed with a fragment from pRS416-INO80-FLAG (Shen et al., 2003) ligated to a URA3 cassette. MEC1 was chromsomally-tagged by insertion of double FLAG sequence with a URA3 cassette floxed by LoxP sites before the coding start site (Wu, unpublished). Following selection of positive clones the URA3 marker was removed by expression of Cre recombinase. Whenever possible, strains were grown in selective media to retain plasmids.

All mec1

strains used in this study also contain the deletion of the ribonucleotide reductase inhibitor gene, SML1, which suppresses the lethality of the mec1

strain. The previously described BY4705 mec1

tel1

strain (Chan et al., 2001) also contains deletions of telomere regulatory genes, RIF1 and RIF2, which prevents senescence in the mec1 tel1

strain.

MALDI-MS and -PSD Analysis of HPLC-Purified Ies4 INO80 complex proteins were separated by HPLC using a Magic 2002 system (Michrom Bioresources, Auburn, CA) with a 1x150 mm, 8 µm 4000 Å PLRP-S column (Michrom). HPLC purified Ies4 was mixed with sinapinic acid matrix (LaserBio Labs, Cedeux, France) on the MALDI target. Spectra were acquired by MALDI-MS on an Applied Biosystems Voyager-DE PRO (Foster City, CA) with post-acquisition processing in Data Explorer v.4.0.0.0 consisting of application of a Gaussian smooth (19 point) as described previously (Person et al., 2003). The protein MS was acquired using the myoglobin_linear parameters in positive ion mode, with internal calibration of the Ies4 protein peak at m/z 13,004. To identify phosphorylated peptides, trypsin-digested Ies4 fragments were analyzed by MALDI-MS in positive ion mode with the linear and reflectron detectors, using ACTH_linear and ACTH_reflectron parameters, respectively. Negative ion spectra were acquired in linear mode using the peptide_linear_negative method parameters. Masses were calculated with MS-Digest in the Protein Prospector suite for a trypsin digest with up to 4 missed cleavages (Clauser et al., 1999). MALDI-PSD fragmentation spectra were collected for all putative phospho-peptides, collecting 400 shots for the first PSD segment at mirror ratio 1.0. This segment is optimized for the mass range containing the parent ion and daughter fragments produced by loss of phosphoric acid.

Supplemental References

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Benjamini, Y., and Hochberg, Y. (1995). Controlling the False Discovery Rate - a Practical and Powerful Approach to Multiple Testing. Journal of the Royal Statistical Society Series B-Methodological 57, 289-300.

Burke, D., Dawson, D., and Stearns, T. (2000). Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual (Cold Spring Harbor: Cold Spring Harbor Laboratory Press).

Chan, S. W., Chang, J., Prescott, J., and Blackburn, E. H. (2001). Altering telomere structure allows telomerase to act in yeast lacking ATM kinases. Curr Biol 11, 1240-1250.

Clauser, K. R., Baker, P., and Burlingame, A. L. (1999). Role of accurate mass measurement (+/- 10 ppm) in protein identification strategies employing MS or MS/MS and database searching. Anal Chem 71, 2871-2882.

Fazzio, T. G., Kooperberg, C., Goldmark, J. P., Neal, C., Basom, R., Delrow, J., and Tsukiyama, T. (2001). Widespread collaboration of Isw2 and Sin3-Rpd3 chromatin remodeling complexes in transcriptional repression. Mol Cell Biol 21, 6450-6460.

Keogh, M. C., Kim, J. A., Downey, M., Fillingham, J., Chowdhury, D., Harrison, J. C., Onishi, M., Datta, N., Galicia, S., Emili, A., et al. (2006). A phosphatase complex that dephosphorylates gammaH2AX regulates DNA damage checkpoint recovery. Nature 439, 497-501.

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